SUMMARY Hereditary spastic paraplegia (HSP) is a large heterogeneous group of neurogenetic disorders caused by the length-dependent degeneration of cortical motor neuron axons. Cortical motor neurons, a group of projection neurons located in motor cortex, control muscle movement through lower motor neurons in the brain stem and spinal cord. Degeneration of these neurons interrupts the signal transmission from brain to spinal cord and then muscles, resulting in progressive spasticity and weakness in muscles. Currently, there remains a lack of effective treatment to ameliorate, stop, or reverse axonal defects in HSPs. Recent studies show that several HSP proteins can regulate the size of lipid droplets, implying their roles in lipid metabolisms. Glial cells play an important for generating and regulating lipid metabolism in the brain. However, whether lipid metabolism is altered in HSP brain and what role glial cells play in the pathogenesis of HSP are largely unknown. The goal of this proposed study is to dissect the novel role of lipid metabolism and the interplay between glial cells and neurons in the pathogenesis of HSP using co-cultures of cortical neurons and glial cells derived from iPSCs of SPG3A patients. SPG3A is the most common early-onset form of HSP caused by mutations in the ATL-1 gene that encodes atlastin-1 protein. We will test our hypotheses by pursuing the following three aims: 1) to identify the contribution of glial cells to axonal and synaptic defects in SPG3A, 2) to determine the role of glial cells in impaired cholesterol homeostasis in SPG3A, and 3) to rescue axonal and synaptic defects in SPG3A by targeting the impaired glia-neuron interaction. By comparing co-cultures of cortical neurons with normal or SPG3A glial cells, our study will provide insights into the role of glial cells in HSP. The cause-effect relationship between atlastin-1 mutations and axonal phenotypes will be confirmed by rescuing the mutations in SPG3A iPSCs and by knocking in mutations to normal human pluripotent stem cells. Moreover, rescue experiments will be performed to identify potential approaches for mitigating axonal and synaptic defects in HSP through regulating lipid metabolism in glial cells. Together, our study is expected to reveal novel roles of glial cells in the pathogenesis of HSP and identify new targets for therapeutic intervention in HSP.